Mycobacterium tuberculosis has the ability to persist for many years in a quiescent form within a human host, only to reactivate and produce communicable disease after the host immune system falters. The quiescent form of the bacteria is believed to be in a state of growth arrest induced by host adaptive immunity. Thus, understanding the mechanisms of metabolic adaptation during the shift between replicative and nonreplicative state will facilitate the development of anti-tuberculosis drugs that target unique metabolic pathways in the persistent state. The goal of this project is to dissect the roles of triacylglycerol (TAG) metabolism in M. tuberculosis persistence and regrowth. The proposal is based on an experiment-derived metabolic model of M. tuberculosis, which is marked with a coordinated carbon flux from key central metabolic pathways for biosynthetic precursor synthesis towards the formation of TAG during establishment of the persistent state. TAG accumulation in M. tuberculosis is proposed to serve as critical carbon and energy fuel for both its long-term persistence and regrowth during activation of the disease. To test the hypothesis, a novel approach is utilized to engineer the TAG metabolic pathways so that the dynamics of TAG metabolism can be manipulated to evaluate its functions during M. tuberculosis dormancy and regrowth. A TAG-deficient M. tuberculosis strain will first be generated by employing an engineering strategy that combines targeted deletion of a major TAG synthase gene with the conditional high expression of a highly active TAG lipase gene. Then, the dynamics of TAG metabolism and roles of TAG on M. tuberculosis survival and re-growth will be assessed in in vitro dormancy models and in a rabbit tuberculosis model. If successful, findings from this project are expected to provide new lines of research focusing on TAG metabolism from which new drugs could be developed to tackle the persistent bacilli and to prevent the disease from reactivation. Moreover, the TAG-deficient M. tuberculosis could be a potential candidate for a tuberculosis vaccine. PUBLIC HEALTH RELEVANCE: The goal of the program is to study the roles of triacylglycerol (TAG) metabolism in Mycobacterium tuberculosis survival during dormancy and bacterial re-growth during disease reactivation. The experimental procedures combine molecular engineering and biochemical/physiological assays using models of M. tuberculosis dormancy in vitro and in vivo. The program is expected to gain new insights on the metabolic adaptation mechanisms of the tubercle bacilli during transition between different growth states and to offer new strategies to tackle the persistent infection and disease reactivation.